1. Field of the Invention
The present invention concerns a novel class of N-substituted N-carboxyanhydride derivatives of .alpha.-amino acids. The compounds of the invention are valuable intermediates or reagents for use in peptide syntheses. The invention also relates to peptide syntheses process using the novel compounds.
2. Prior Art
The synthesis of peptides involves the establishment of amide bonds by the condensation of the carboxyl group of one amino acid with the amino group of another. In order to achieve the desired sequence and to avoid polycondensation, it is necessary to protect the functional groups which are not intended to react, i.e. the amino group of the carboxyl component and the carboxyl group of the amino component, respectively, as well as possible side-chain functional groups. All protecting groups must be sufficiently labile to allow quantitative removal without concomitant cleavage of peptide bonds or any other kind of destruction.
Shortly before the turn of the century, the first attempts at a stepwise assembly of amino acids into peptides were made. The lack of a selectively removable amino protecting group, however, imposed severe limitations upon the results which could be obtained at that time. This difficulty was not overcome until 1932, when the benzyloxycarbonyl group, which can be removed selectively by catalytic hydrogenation, was introduced.
In the decades where were to follow, a great number of smaller peptides were synthesized, and from 1950 to 1960, the first entirely synthetic peptide hormones, chemically and biologically identical with the natural ones, were produced.
The very principle of the synthesis remained practically unchanged throughout this period, but several improvements were introduced into the classical synthesis, such as more suitable types of derivatives and more varied ways of condensation; especially worthy of mention is the now most widely used method, employing tert.butoxycarbonyl for amino protection and N,N'-dicyclohexylcarbodiimide as activating reagent fr condensing agent.
In the classical peptide synthesis, the reaction proceeds in solution, where equivalent quantities of amino and carboxyl components are reacted, influenced by the activating reagent, e.g. N,N'-dicyclohexylcarbodiimide. At the end of the reaction, the product is isolated by precipitation, but due to co-precipitation of starting material and side-products, a time-consuming purification must usually be carried out. Thus, in the case of longer-chain peptides, recrystallization or re-precipitation is mostly insufficient, and one has to take recourse to counter-current distribution, gel-filtration or ion-exchange chromatography. This procedure is frequently very tedious, and yields of from 40% to 60% of the theoretical in fragment condensation, and from 60% to 90% in stepwise chain elongation are usually regarded as satisfactory.
In 1962, a novel concept, eliminating all solubility problems by rendering the growing peptide chain insoluble by covalent bonding to a resin matrix, was introduced (the so-called solid-phase peptide synthesis). Here, the washing-out of excess amino acid derivative and by-products can be carried out quantitatively with adequate volumes of various solvents without less of any of the resin-bound peptide. Moreover, the resin-bound peptide can be isolated simply by filtration, a fact which makes the process amenable to automation.
The principle of the solid-phase peptide synthesis is exemplified below in schematic form, t-BOC representing the tert.butoxycarbonyl group. ##STR1##
In the solid-phase synthesis, the growing peptide chain is constantly anchored by ist C-terminus to a resin, usually co-valently bound in the form of a benzyl ester to an insoluble styrene-divinylbenzene co-polymer. In each cycle of the synthesis, dissolved amino acid derivative is added, followed by the condensing agent, usually N,N'-dicyclohexylcarbodiimide. Notwithstanding the two-phase nature of the reaction, it proceeds quickly, due to efficient mixing and the pronounced swelling of the resin in the solvent used. Traditionally, the .alpha.-amino group of the amino acid is protected by the tert.butoxycarbonyl group, which, unlike the benzyloxycarbonyl group, is readily split off upon brief exposure to N hydrogen chloride in glacial acetic acid or dioxane after formation of the amide bond. When the peptide has attained the desired chain-length, the bonds to the resin and to the N-terminal and side-chain protecting groups are cleaved by briefly passing dry hydrogen bromide gas through a suspension of the peptide-resin in anhydrous trifluoroacetic acid. The liberated peptide is dissolved, and can be isolated as the hydrobromide. The removal of certain side-chain protecting groups does, however, require further treatment, which will not be dealt with here.
Recently, other methods of peptide synthesis have been developed, in which the growing peptide chain is anchored to a soluble resin; in principle, these syntheses offer the same technical advantages as the solid-phase synthesis, the actual method of effecting the separation being different, e.g. gel-filtration. In the following text, peptide syntheses of this kind and solid-phase syntheses will be referred to collectively as syntheses using polymeric carriers, or briefly, resin carrier syntheses.
Due to the danger of formation of peptides containing a wrong sequence because of incomplete reaction in certain steps of the resin carrier synthesis, a suitable excess of amino acid derivative is used, in general from two to three times the theoretical quantity, in order to make each step proceed to completion.
Certain activated amino acid derivatives are particularly prone to suffer rearrangement into inactive products. In consequence, the remaining quantity of derivative may be inadequate to ensure a quantitative reaction. A generally occurring rearrangement in carbodiimide syntheses is illustrated schematically below: ##STR2##
This rearrangement is especially pronounced in the case of glycine, and for this reason an excess of this amino acid is usually employed, in resin carrier syntheses generally five times the theoretical amount.
A further, marked disadvantage attending the use of activating reagents like e.g. dicyclohexylcarbodiimide is their high degree of reactivity, which requires an extensive protection of side chain functional groups, often a complete or so-called "global" protection. Furthermore, the interaction of the activating reagent and the N-protected amino acid produces an activated complex, the excess of which may well be isolated after the condensation, but cannot be utilized to regenerate the N-protected amino acid. In practice, therefore, this means that the excess of N-protected amino acid--a most expensive reagent--employed in each condensation step is lost. Even in classical syntheses in solution this is a serious drawback, because, due to the abovementioned rearrangement, an excess of the N-protected derivative must often be used. In resin carrier syntheses this drawback assumes an even greater significance, since in order to achieve the very essential acceleration of the synthetic procedure, one has to acquiesce in the sacrifice of the large excess of N-protected amino acid, which especially in industrial-scale syntheses represents a great financial loss.
For this reason, there is a great current interest in finding other types of reagents and condensation methods which are not connected with disadvantages of the nature mentioned above. One solution suggested in this connection is the use of activated esters of amino acids, since the employment of such activated esters is not dependent upon the presence of added condensing agent, and consequently, no undesired conversion of the excess reagent takes place. Activated esters, among which especially p-nitrophenyl-, pentachlorophenyl-, and N-hydroxysuccinimide esters have found practical application, are, however, very reactive species, and particularly in the case of p-nitrophenyl and pentachlorophenyl esters, difficulties have been experienced in their preparation and storage. Moreover, such activated esters have often been found to react sluggishly in resin carrier syntheses.
A particularly interesting type of N-protected, reactive derivatives of .alpha.-amino acids for use in peptide syntheses are the so-called N-carboxyanhydrides of .alpha.-amino acids (amino acid NCAs), most of which can be prepared by the action of phosgene upon .alpha.-amino acids, and which are represented by the formula ##STR3## where R designates the side-chain of the amino acid. In these N-carboxyanhydrides, the anhydride moiety has a double role, namely that of activating the carboxyl function as well as protecting the amino function. In syntheses employing N-carboxyanhydrides, no additional condensing agent is used, whereby side-reactions with unprotected amino acid side-chains, occasioned by the presence of such agents, are avoided. Thus it has been reported that only the side-chain functions of lysine and cysteine need be protected. At the same time, a conversion of the added amino acid derivative into an activated form, which precludes the recovery of the derivative, is avoided.
The fact that the N-carboxyanhydride method, in spite of the abovementioned advantages, has not gained any widespread application, is due to a number of factors. One disadvantage of the N-carboxyanhydrides is their relative instability during storage; if a ring-opening occurs in just a small fraction of the N-carboxyanhydride molecules, the free amino groups formed will react with the activated carboxyl groups of other N-carboxyanhydride molecules, resulting in a steadily increasing polymerization by chain reaction, noticeable e.g. by a steady increase in the melting point. Under unfavourable storage conditions, this reaction may be complete in just a few days. Furthermore, for the same reason, special protective measures must be taken during the handling of N-carboxyanhydrides due to their reactivity, such as the exclusion of humidity by the use of e.g. a glove-box. Moreover, in model experiments N-carboxyanhydrides have been shown to react with the "wrong" side of the anhydride, i.e. with the carbonyl group attached to the nitrogen atom, so that by the reaction with the amino group of the amino component a carbonyl group is inserted between the two nitrogen atoms, forming an ureido acid. The more basic the amino component, the more extensive the formation of ureido acid. In resin carrier synthesis, where a large excess of acylating amino acid derivative is used, conventional N-carboxyanhydrides are useless, because the free amino group of the peptide formed will react further with excess N-carboxyanhydride. Such N-carboxyanhydrides have therefore only been useful in peptide synthesis in solution, and even then mostly in aqueous solution, in which the intermediately formed peptide carbamate can be stabilized by strict adherence to certain conditions of pH and temperature for the short duration of the condensation reaction.
In an attempt to circumvent the abovementioned problems attending the otherwise attractive use of N-carboxyanhydrides, Block and Cox, cf. "Peptides, Proc. 5th Europ. Symp., Oxford September 1962", Pergamon Press 1963, Ed. G. T. Young, pp. 84-87, tried to prepare N-trityl-N-carboxyanhydrides of .alpha.-amino acids, but were only able to synthesize these derivatives of the simplest .alpha.-amino acids, viz. glycine and alanine. The same workers tried to prepare N-substituted N-carboxyanhydrides in which the N-substituent was benzyloxycarbonyl and tert.butoxycarbonyl, but reported that such derivatives could not be made. Apart from the fact that it was impossible to prepare the N-trityl derivatives of the N-carboxyanhydrides of other than the two simplest .alpha.-amino acids, it is common knowledge that N-trityl amino acids in various condensation methods of peptide synthesis produce low yields in the stepwise method of chain elongation, due to the considerable sterical hindrance imposed by the trityl group upon the carboxyl group of the attached amino acid. Thus, notwithstanding the results published in 1963 and the suggestion by Hanson and Law in 1965, cf. J. Chem. Soc. 1965, pp. 7284-7297, to use various methoxy-substituted benzhydryl groups as N-protecting groups for N-carboxyanhydrides, the work of recent years has mainly been directed at the use of unsubstituted N-carboxyanhydrides under closely controlled conditions, cf. e.g. Hirschmann et al. "The Controlled Synthesis of Peptides in Aqueous Medium. VIII. The Preparation and Use of Novel .alpha.-Amino Acid N-Carboxyanhydrides" , J. Amer. Chem. Soc. 93:11, June 1971, pp. 2746-2754.
Considering the attractive aspects involved, the reason for the lack of progress in the known art development of N-carboxyanhydrides of .alpha.-amino acids carrying an N-substituent, which after the participation of the N-carboxyanhydride in the condensation reaction serves to protect the amino group of the resulting peptide against further reaction, may presumably be attributed to the fact that such N-substituted N-carboxyanhydrides must meet every condition in a very critical combination of conditions, namely:
(1) The N-substituted N-carboxyanhydrides in question should be reasonably easy to prepare, PA0 (2) they should be stable, so as not to require special precautions in their storage, PA0 (3) the derivatives should crystallize well, so as to facilitate isolation and characterization, PA0 (4) the sterical hindrance exerted by the N-substituent upon the carboxyl group of the attached amino acid should be very small, in order that the condensation reaction may be complete within a reasonable time, PA0 (5) the substituent in question should effectively protect the terminal amino group of the peptide prepared by the action of the N-carboxyanhydride, but on the other hand it should PA0 (6) be easily removable from the peptide under conditions not conducive to damage even to sensitive peptide bonds, when the terminal amino group is to be made available for further reaction.